The overall objective of this revised competitive renewal application is to determine the molecular mechanisms responsible for Hyperhomocysteinemia (HHcy)-induced endothelial cell (EC) growth inhibition. We provided initial evidence demonstrating that pathophysiologically relevant concentrations of homocysteine (Hcy) inhibit EC growth, but not that of other cell types, through a hypomethylation related mechanism. In the previous grant period, we discovered that Hcy inhibits cyclin A transcription, DNA methyltransferase 1 (DNMT1) activity and DNA methylation in cyclin A promoter, and that adenovirus-transduced expression of cyclin A and DNMT1 genes rescued EC growth from the inhibitory effect of Hcy. Our in vivo studies indicate that HHcy impairs endothelial function and eNOS activity via PKC activation, and that HHcy impaired reendothelialization and increased neointimal formation in mice. Our basic hypothesis is that that Hcy impairs reendothelialization via inhibition of EC proliferation, and contribute, to the increased atherosclerosis in HHcy. This project will study this hypothesis utilizing three linked specific aims. First, in Aim 1, we will explore the regulatory mechanisms of Hcy-induced cyclin D2/D3 suppression in EC. Second, in Aim 2, we will Determine biochemical basis of Hcy-hypomethylation and growth inhibition in EC. Finally, in Aim 3, we will examine the effect of EC therapy in post-injury reendothelialization and neointima formation in HHcy mice. We believe that completion of the specific aims should provide valuable new information to establish the links between HHcy and atherosclerosis, and lead to therapeutic advantage. PUBLIC HUMAN RELEVANCE It has been suggested that HHcy accounts for the higher prevalence of CVD in renal disease, diabetes, ageing and in postmenopausal women that is not explained by traditional risk factors. However, the underlying mechanism is largely unknown and the role of homocysteine (Hcy)-induced endothelial growth inhibition in CVD is unclear. We have previously demonstrated that Hcy exerts highly selective inhibitory effect on cyclin A transcription and EC growth through a hypomethylation related mechanism, which blocks cell cycle progression and endothelium regeneration. Since endothelial injury is an early event in vascular disease, and since endothelial regeneration determines the onset of atherosclerosis, we hypothesize that Hcy promotes atherosclerosis by impairing reendothelialization via inhibition of EC proliferation. In this proposal, we propose to investigate the role and mechanisms of HHcy in altering EC metabolism and EC biology, with the goal of identifying the underlying mechanisms, using in vitro and in vivo approaches.